Throughout this application various publications are referenced within parentheses. The disclosures of these publications in their entireties are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains.
The present invention relates generally to apoptosis or, programed cell death, and more particularly, nucleic acids encoding human Bad polypeptides which can be used to modulate apoptosis for the therapeutic treatment of human diseases.
Apoptosis is a normal physiological process of cell death that plays a critical role in the regulation of tissue homeostasis by ensuring that the rate of new cell accumulation produced by cell division is offset by a commensurate rate of cell loss due to death. It has now become clear that disturbances in apoptosis, also referred to as physiological cell death or programmed cell death, that prevent or delay normal cell turnover can be just as important to the pathogenesis of diseases as are known abnormalities in the regulation of proliferation and the cell cycle. Like cell division, apoptosis is similarly regulated under normal circumstances through complex interaction of gene products that either promote or inhibit cell death.
The stimuli which regulate the function of these apoptotic gene products include both extracellular and intracellular signals. Either the presence or the absence of a particular stimuli can be sufficient to evoke a positive or negative apoptotic signal. For example, physiological stimuli that prevent or inhibit apoptosis include, for example, growth factors, extracellular matrix, CD40 ligand, viral gene products neutral amino acids, zinc, estrogen and androgens. In contrast, stimuli which promote apoptosis include growth factors such as tumor necrosis factor (TNF), Fas, and transforming growth factor xcex2 (TGFxcex2), neurotransmitters, growth factor withdrawal, loss of extracellular matrix attachment, intracellular calcium and glucocorticoids, for example. Other stimuli, including those of environmental and pathogenetic origins, also exist which can either induce or inhibit programmed cell death. Thus, apoptosis is mediated by diverse signals and complex interactions of cellular gene products which ultimately result in a cell death pathway that is required to regulate normal tissue homeostasis.
Several gene products which modulate the apoptotic process have now been identified. Although these products can in general be separated into two basic categories, gene products from each category can function to either inhibit or induce programmed cell death. One family of gene products are those which are members of the Bcl-2 family of proteins. Bcl-2, is the best characterized member of this family and inhibits apoptosis when overexpressed in cells. Other members of this gene family include, for example, Bax, Bak, Bcl-xL, Bcl-xs, and Bad. While some of these proteins can prevent apoptosis, others such as Bax, Bcl-xs and Bak function to augment apoptosis.
A second family of gene products which modulate the apoptotic process is the family of proteases known as aspartate-specific cysteine proteases (ASCPs). These proteases are related genetically to the ced-3 gene product which was initially shown to be required for programmed cell death in the roundworm, C. elegans. The ASCPs family of proteases includes, for example, human ICE (interleukin-1-xcex2 converting enzyme), ICH-1L, ICH-1S, CPP32, Mch2, Mch3, ICH-2 and ICErelxe2x88x92III. One common feature of these gene products is that they are cysteine proteases with specificity for substrate cleavage at Asp-X bonds. Although these proteases induce cell death when expressed in cells, several alternative structural forms such as ICExcex4, ICExcex5, ICH-1S and Mch2xcex2 are known which actually function to inhibit apoptosis.
In addition to the Bcl-2 and ASCP gene families which play a role in apoptosis in mammalian cells, it has become increasingly apparent that other gene products exist which are important in mammalian cell death and which have yet to be identified. For example, in addition to Ced-3, another C. elegans gene known as Ced-4 exists which is also required for programmed cell death in C. elegans. However, mammalian homologues of this protein remain elusive and have not yet been identified. Further, it is ambiguous as to whether other genes exist which belong to either of the above two apoptotic gene families or what role they may play in the programmed cell death pathway. Finally, it is also unclear what the physiological control mechanisms are which regulate programmed cell death or how the cell death pathways interact with other physiological processes within the organism.
The maintenance of tissue homeostasis is required in a range of physiological processes such as embryonic development and immune cell regulation as well as in normal cellular turnover. The dysfunction, or loss of regulated apoptosis can lead to a variety of pathological disease states. For example, the loss of apoptosis can lead to the pathological accumulation of self-reactive lymphocytes such as that occurring with many autoimmune diseases. Inappropriate loss of apoptosis can also lead to the accumulation of virally infected cells and of hyperproliferative cells such as neoplastic or tumor cells. Similarly, the inappropriate activation of apoptosis can also contribute to a variety of pathological disease states including, for example, acquired immunodeficiency syndrome (AIDS), neurodegenerative diseases and ischemic injury.
Treatments which are specifically designed to modulate the apoptotic pathways in these and other pathological conditions can change the natural progression of many of these diseases. However, since distinct differences are known to exist between human and other species such as C. elegans, it is advantageous to use human proteins for the production of biopharmaceuticals as well as other molecules or compounds which are produced to modify the function of a regulator of programed cell death. The use of human proteins as both biopharmaceuticals and to identify compounds is also important in regard to the therapeutic efficacy and safety of the treatment. For example, the half-life of non-human proteins can be comprised in a heterologous environment and the induction of a host immune response against a non-human protein can be potentially dangerous to the patient.
Thus, there exists a need to identify new apoptotic genes and their human homologues and for methods of modulating apoptotic process for the therapeutic treatment of human diseases. The present invention satisfies this need and provides related advantages as well.
The invention provides an isolated gene and an isolated nucleic acid sequence encoding human Bad and functional fragments thereof. Also provided is an isolated human Bad polypeptide and functional fragments thereof. Methods of identifying human Bad binding partners and methods of screening for compounds which interfere with the association of human Bad interacting polypeptides with human Bad are also provided. Finally, methods for decreasing or increasing the viability of a cell are provided as well.